Exploration of a natural product's mode of action often sheds light on basic areas of biology not easily studied by traditional approaches. This bioorganic strategy to cell biology has led to significant advances in the study of immunoregulation using the natural products, FK506, cyclosporin, and rapamycin. In addition, the investigation of how potent biologically active natural products work at the molecular level (through the identification of their intracellular protein receptors), can lead to the identification of key proteins within a complex intracellular process. These natural product target proteins are, by definition, 'pharmaceutically vulnerable' and thus can serve as novel drug targets. This proposal focuses on the modes of action of two natural products; the anti-inflammatory fungal metabolite isopanepoxydone and the potent antitumor macrolide amphidinolide B isolated from an Okinawan dinoflagellate. Isopanepoxydone blocks the DNA binding activity of NF-kappaB, a key transcription factor that mediates pro-inflammatory signal-induced gene transcription. This is mediated via inhibition of I?B degradation, a protein that negatively regulates the activity of NF-kappaB. Although much is known about the downstream consequences of isopanepoxydone's anti-inflammatory action, the protein target of this natural product that lies upstream of IkappaB is unknown. Towards the goal of identifying the intracellular target of isopanepoxydone, we have modified our previously reported synthetic route to generate a biotinylated isopanepoxydone affinity reagent. Here, we present evidence for a 48kDa protein that covalently and specifically binds to this isopanepoxydone affinity reagent. The objective of this research is to understand the molecular mechanisms by which isopanepoxydone inhibits pro-inflammatory signal transduction through the purification, identification and characterization of this 48kDa isopanepoxydone binding protein. The anti-tumor macrolide amphidinolide B is at an earlier stage in the mode of action elucidation process. We propose here a novel retrosynthetic disconnection strategy for the total synthesis of this potent natural product. This synthetic effort will also afford the facile generation of a biotinylated affinity reagent, with which we plan to purify, identify and clone amphidinolide B binding proteins. Biochemical and cell biological characterization of these amphidinolide binding proteins will confirm their role in mediating the potent cytotoxicity of the natural product. We have successfully employed this same chemical/biochemical/cell biological approach to identify and characterize the intracellular receptors of the antiangiogenic natural product, fumagillin, the anti-inflammatory agent, parthenolide, and the antitumor compounds, epoxomicin and eponemycin.